This invention relates to a method of carbohydrate engineering of glycoproteins. More particularly, the present invention concerns a method of modifying the carbohydrate moiety on mammalian cell-secreted glycoproteins to facilitate the structural and functional analysis of the secreted glycoproteins.
A large number of proteins of key scientific and medical interest are heavily glycosylated. In many cases the carbohydrate accounts for 50% of the molecular weight of the glycoprotein. This can present serious obstacles to the structural analysis of these molecules by crystallographic and NMR-based procedures. As discussed previously (Davis et al., 1993), the oligosaccharides may obscure the protein surface, or oligosaccharide microheterogeneity (the presence of multiple glycoforms) may prevent the formation of reproducible crystal contacts involving the oligosaccharides directly. Alternatively, if the oligosaccharides are able to form crystal contacts, the flexibility and mobility of the oligosaccharides may limit the order of the crystals. In the case of NMR analyses, heavy glycosylation may increase spectral complexity, exacerbate peak broadening due to increased molecular weight, or impose peak microheterogeneity as a result of variation in the length and composition of the oligosaccharides present on each of the glycoforms.
Although a detailed survey has not been published, in the experience of the present inventors the removal of complex oligosaccharides from glycoproteins is generally difficult. Several approaches for preventing the addition of oligosaccharides or for facilitating their removal may be successful in individual cases; however, each of these approaches has significant limitations so that general solutions to the problem of glycosylation have been elusive (discussed in Davis et al., 1993). The ideal strategy is one that allows (1) the normal transfer of the Glc.sub.3 Man.sub.9 GlcNAc.sub.2 oligosaccharide precursor to the protein and therefore the correct folding of the glycoprotein in the endoplasmic reticulum of eukaryotic cells and (2) the subsequent inhibition of oligosaccharide processing to complex forms, thus rendering the oligosaccharides endo H-sensitive and allowing their subsequent removal prior to structural analysis.
The effectiveness of such a strategy has been demonstrated by producing the cell adhesion molecule, rat soluble CD2 (sCD2), in a Chinese Hamster ovary (CHO) cell glycosylation mutant, Lec3.2.8.1 (Stanley, 1989), which is largely defective in processing oligosaccharides beyond endo H-sensitive, Man.sub.5 GlcNAc.sub.2 forms (Davis et-al., 1993). This work led directly to a crystal structure for rat sCD2 (Jones et al., 1992). However, this approach suffers from the drawbacks that the Lec3.2.8.1 cell line is difficult to transfect (Davis et al., 1993) and yields of fully endo H-sensitive glycoforms can in some instances be less than 50% (S. J. Davis, unpublished).
Further background information on the analysis of secreted and membrane-associated glycoproteins can be had by reference to the recent review article by Dwek et al., Ann. Rev. Biochem. 62. 65-100 (1993).